Two growth modulating proteins have originally been characterized by their ability to reversibly induce phenotypic transformation of mammalian cells in vitro and have therefore been designated as Transforming Growth Factors type .alpha. and type .beta. (Anzano, M. A. et al. (1983) PNAS 80, 6264-6268). Despite their common nomenclature TGF-.alpha. and TGF-.beta. have shown to be both structurally as well as functionally entirely distinct proteins each acting through its own unique receptor system. TGF-.alpha. which competes with epidermal growth factor (EGF) for binding to the same cell surface receptor (Todaro, G. J. et al. (1980) PNAS 77, 5258-5262) and which shares sequence homologies and similar activity with EGF (Marquardt, H. et al. (1984) Science 223, 1079-1082) is synthesized as a transmembraneous precursor of 159 amino acids and is proteolytically processed into a peptide of 50 amino acid residues (Derynck, R. et al. (1984) Cell 38, 287-297). As a potent mitogen for mesenchymal cells, TGF-.alpha. is produced and released by numerous transformed cell lines and human cancers, but is also expressed in activated macrophages and in other normal tissues, thus making its role in neoplasia still unclear.
TGF-.beta. was originally purified to homogeneity from human platelets (Assoian, R. K. et al. (1983) J. Biol. Chem. 258, 7155-7160), human placenta (Frolik, C. A. et al. (1983) PNAS 80, 3676-3680) and bovine kidney (Roberts, A. B. et al. (1983) Biochemistry 22, 5692-5698) and identified as a homodimeric protein with a molecular mass of 25.000 D. First characterized by its ability to act synergistically with EGF or TGF-.alpha. to induce anchorage-independent growth of untransformed NRK cells, recently, TGF-.beta. has been shown to exhibit numerous regulatory effects on a wide variety of both normal and neoplastic cells indicating the importance of this protein as a multifunctional regulator of cellular activity. TGF-.beta. may either stimulate mitogenesis, cell proliferation and growth, or may effectively inhibit said processes, or may exhibit other actions like e.g. control of adipogenesis, myogenesis, chondrogenesis, osteogenesis und immune cell function, stimulation of chemotaxis, or induction or inhibition of differentiation depending upon the cell or tissue type, and the presence or absence of other growth factors. Many of the actions of TGF-.beta. are related to the response of cells or tissues to stress or injury, and to the repair of resultant damage. After inflammation, TGF-.beta. plays the major role in the formation of granulation tissue, increases the expression of genes associated with extracellular matrix formation such as flbronectin, collagen and several protease inhibitors and stimulates collagen-matrix contraction by fibroblasts, suggesting its possible role in connective tissue contraction (Roberts, A. and Sporn, M. B. (1988) Adv. Cancer Res. 51, 107-145; Sporn, M. B. and Roberts, A. (1989) J. Amer. Med. Assoc. 262, 938-941).
Until now, three distinct types of TGF-.beta.s designated as TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3 which are functionally closely related and share a high degree of receptor cross-reactivity have been cloned and characterized by sequence analysis. All TGF-.beta.s are synthesized as 390 to 412 amino acid precursors that undergo proteolytic cleavage to produce the monomeric forms, which consist of the C-terminal 112 amino acids. In their mature, biologically active forms, TGF-.beta.s are acid- and heat-stable disulfide-linked homodimers of two polypeptide chains of 112 amino acids each. The complete amino acid sequences of human (Derynck, R. et al. (1985) Nature 316, 701-705), murine (Derynck, R. et al. (1986) J. Biol. Chem. 261, 4377-4379) and simian TGF-.beta.1 (Sharples, K. et al. (1987) DNA 6, 239-244) show remarkable sequence conservation, differing only in a single amino acid residue. Comparison of the amino acid sequence of human TGF-.beta.1, human TGF-.beta.2 (de Martin, R. et al. (1987) EMBO J. 6, 3673-3677; Marquardt, H. et al. (1987) J. Biol. Chem. 262, 12127-12131) and human TGF-.beta.3 (Ten Dijke, P. et al. (1988) PNAS 85, 4715-4719) has demonstrated that the three proteins exhibit in their mature forms about 70-80% sequence identity. A heterodimeric TGF-.beta.1.2 has been isolated from porcine platelets and consists of one subunit of TGF-.beta.1 disulfide-linked to one subunit of TGF-.beta.2 (Cheifetz, S. et al. (1987) Cell 48, 409-415).
Recently, attempts have been undertaken aiming to produce TGF-.beta.s by means of recombinant techniques rather than isolating these factors from natural sources (e.g. platelets) in order to obtain sufficient amounts for testing in various therapeutic modalities. However, it has proven to be extremely difficult to synthesize recombinant TGF-.beta. while retaining its biological activity. As can be seen from the sequences depicted in the sequence listing under SEQ ID No. 1, 2, and 3 and 41, 42 and 43, the 112 amino acids containing mature forms of TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3 contain 9 cysteine residues each, at least some of which are involved in intrachain and interchain disulfide bond formation which results in the complex tertiary structure of the biologically active, dimeric molecules. Heterologous expression of TGF-.beta. may lead to a product which, although having the correct primary structure, fails to fold properly to produce the correct secondary or tertiary structures and which, therefore, lacks the biological activity. To date, the secondary and tertiary structures of TGF-.beta.s are unknown.
Taking the complexity of the native TGF-.beta. molecules into account, it has generally been considered expedient to express the respective TGF-.beta. genes in cells derived from higher organisms. The expression of simian and human TGF-.beta.1 in Chinese hamster ovary (CHO) cells under the control of the SV40 promoter is described in European Patent Applications 293,785 and 200,341, respectively. Recombinant TGF-.beta.2 could be expressed in the same cell line as disclosed in European Patent Application 268,561 and in German Offenlegungsschrift 38 33897. Eukaryotic expression of a fusion protein of TGF-.beta.3 (with TGF-.beta.1) is disclosed in European Patent Application 267,463.
Although expression of recombinant TGF-.beta.s can be achieved in eukaryotic systems, the yields of biologically active, correctly folded material obtained are still far from being satisfactory. On the other hand, it seemed unlikely that biologically active TGF-.beta. could be obtained when the respective gene was expressed in a microbial host, since in e.g. bacteria the intracellular conditions are not conducive to refolding, disulfide bond formation and disulfide-stabilized dimerization which is apparently essential for activity. Thus, only very little biologically active TGF-.beta.2 could be obtained after expression of the respective gene in E. coli under the control of the lambda promoter as described in European Patent Application 268,561. This lack of activity is considered to be due to the fact, that the biologically active, dimeric form of TGF-.beta.2 fails to form spontaneously from the monomeric primary translation product when exposed to the reducing environment inside the bacterial cells. Another report describes the expression of TGF-.beta. cDNA in E. coli under the control of the trp promoter yielding a radioactively labelled protein band with an apparent molecular weight of 13,000 D in an autoradiogram of a SDS polyacrylamide gel, but no activity was measured (Urushizaki, Y. et al. (1987) Tumor Res. 22, 41-55).
When recombinant proteins are produced at high levels in bacterial (such as E. coli) expression systems, they often appear in the form of highly insoluble intracellular precipitates referred to as inclusion bodies or refractile bodies (Brems, D. N. et al. (1985) Biochemistry 24, 7662) which can be recognized as bright spots visible within the enclosure of the cells under a phase contrast microscope at magnifications down to 1000 fold. These inclusion bodies, which can readily be separated from the soluble bacterial proteins, contain the recombinant protein in a mostly denatured and reduced form which does not exhibit the functional activity of its natural counterpart and which therefore is useless as a commercial product.
It is therefore generally agreed, that the recombinant refractile protein has to be solubilized under conditions which are suitable in maintaining it in its denatured form and subsequently has to be refolded in order to undergo the transition from the denatured unfolded form to the proper, functionally active three-dimensional structure, the conformation of which is stabilized by relatively weak interatomic forces such as hydrogen bonding, hydrophobic interactions and charge interactions. In the case of cysteine containing proteins this process may also involve formation of disulphide bonds. When the formation of disulfide bonds is chemically promoted, the formation of incorrect intramolecular and, in the case of dimeric or multimeric proteins, intermolecular bridges should be prevented or at least minimized, since the formation of undesired, incorrectly folded isomers may yield non-homogenous material, thus complicating the further purification of the protein having the desired structure, or may generate a protein with reduced activity.
A number of publications have appeared which report refolding attempts for individual proteins produced in bacterial hosts, or which are otherwise in a denatured or non-native form. Formation of a dimeric, biologically active human colony stimulating factor-1 (CSF-1) after expression in E. coli is described in PCT Application No. 88/8003 and by Halenbeck, R. et al. (1989) Biotechnology 7, 710-715. The procedures described involve the steps of initial solubilization of CSF-1 monomers isolated from inclusion bodies under reducing conditions in a chaotropic environment comprising urea or guanidine hydrochloride, refolding which is achieved by stepwise dilution of the chaotropic agents, and final oxidation of the refolded molecules in the presence of air or a redox-system. In PCT Application No. 88/8849 a process for recovering recombinant interleukin-2 (IL-2) is disclosed, characterized in that IL-2 isolated from refractile bodies is denatured under reducing conditions with 6M guanidine hydrochloride, the soluble IL-2 is oxidized by a controlled oxidization in the presence of Cu.sup.2+ ions, and the oxidized IL-2 is refolded by reducing the concentration of the denaturant in the solution. Interleukin-2 and interferon-.mu. (IFN-.beta.) have been refolded using SDS for solubilization and Cu.sup.2+ ions as oxidation promoters of the fully reduced proteins (U.S. Pat. No. 4,572,798). The process for isolating recombinant refractile proteins as described in U.S. Pat. No. 4,620,948 involves strong denaturing agents to solubilize the proteins, reducing conditions to facilitate correct folding and denaturant replacement in the presence of air or other oxidizing agents to reform the disulfide bonds. The proteins to which said process can be applied include urokinase, human, bovine and porcine growth hormone, interferon, tissue-type plasminogen activator, FMD coat protein, prorennin and the src protein. A method for renaturing unfolded proteins including cytochrome c, ovalbumin and trypsin inhibitor by reversibly binding the denatured protein to a solid matrix and stepwise renaturing it by diluting the denaturant is disclosed in PCT Application No. 86/5809. A modified monomeric form of human platelet-derived growth factor (PDGF) expressed in E. coli is S-sulfonated during purification in order to protect thiol moieties and is dimerized in the presence of oxidizing agents to yield the active protein (Hoppe, J. et al. (1989) Biochemistry 28, 2956).
The foregoing references are merely representatives of a huge amount of literature dealing with the refolding of non-native proteins derived from different sources. The man skilled in the art on the other hand knows that the success of refolding experiments cannot be predicted. Unsuccessful experiments are usually not reported. There is no certainty that anyone of the reported refolding conditions would work at all with a given denatured protein such as TGF-.beta.. Considering the fact, that TGF-.beta. is a dimeric protein containing 9 cysteine residues per chain and a number of intramolecular as well as intermolecular disulfide bonds, which are required for activity, it is a particularly difficult challenge to produce biologically active TGF-.beta. from its monomeric, denatured or otherwise non-native form. Nowhere in the literature is a specific process described for the preparation of biologically active dimeric TGF-.beta. from its non-native form.